A program addressing three major functional properties of cytochrome c is outlined. (1) Regulation of cytochrome c structure and functional properties by pH. (a) The pH-dependence of cytochrome c reduction potentials is highly species dependent. Potentiometric, electrochemical, and NMR characteristics of mutants designed to test possible origins of this species variation will be undertaken. (b) Although formation of an alkaline conformation of cytochrome c with a pK~8.5-9 is well known and now appears to be a physiological function of cytochrome c, the mechanism of this conformational change and the active site structure of alkaline cytochrome c are poorly understood. Mutant cytochromes with altered alkaline conformations will be studied by kinetic, EPR, NIR-MCD, and NMR methods, and the functional properties of mutant cytochromes altered at residues critical to the redox-linked change in cytochrome c conformation will be further characterized. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) Potentiometric and NMR methods will be used to study binding of cytochrome c to cytochrome b5 and cytochrome c peroxidase and, in part, binding of cytochrome b5 to hemoglobin. The potentiometric technique provides previously unavailable information concerning complexation-linked changes in proton binding and precise equilibrium constants for complex formation. (b) Specifically modified wild-type and mutant cytochrome c derivatives modified at specific Lys residues to permit their resolution by NMR will be used to identify residues involved in protein-protein recognition. (c) Anaerobic stopped- flow kinetics will be used with Brownian dynamics calculations to study the bimolecular electron transfer between pairs of heme proteins to assess the relative contributions of electrostatic and thermodynamic considerations to the observed rates. (d) Mutants of both proteins modified at the cytochrome c-cytochrome c peroxidase protein-protein interface will be studied by kinetic analysis of various forms of the cytochrome c-(Zncytochrome c peroxidase) complex. (3) New strategies for incorporation of electron donor/acceptor centers in cytochrome c. Two methods for creating a second redox-active sites in cytochrome c are proposed. These involve (a) covalent attachment of flavins through alkylation of mutant surface Cys residues and (b) reconstruction of the environment surrounding Cys-102 in yeast cytochrome c to create a (type I(blue)) copper center.